Recent clinical trials establish that certain rare stem, progenitor or immune cell populations have clinical utility in regenerative medicine and immunotherapy (references). Such rare target cells may be as much as five orders of magnitude (105) less numerous than the other cells in blood or bone marrow, the most numerous of which are red blood cells and platelets. The admixture of abundant unwanted cells with rare, clinically important target cells presents a challenge in enriching and purifying the target cells. The major cell populations in blood and bone marrow differ in size and density, so that stratifying cells based on their densities during centrifugation can be utilized in the selection and purification process. For example, normal human blood generally comprises red blood cells (“RBCs”), the most numerous and most dense cell; platelets (“PLTs”), the smallest and least dense cell; white blood cells (“WBCs”), the largest cells, with a density between the RBCs and the PLTs, and plasma. These three cell fractions separate into distinct populations during centrifugation. On average, RBCs make up approximately 99.9% of an individual's total blood cells and approximately 45% of the total volume of blood within an individual, although this is known to vary among individuals and, over time, within the same individual. RBCs serve a vital function as the principal means of delivering oxygen to body tissues, but are not useful for regenerating tissue or enhancing the immune system to combat certain diseases, including cancers. Nearly all of the remainder of an individual's blood volume is made up of plasma, a non-cellular liquid accounting for approximately 55% of the total blood volume and in which all blood cells are suspended. Thus, over 99% of the volume of normal blood is made up of plasma and RBCs.
The remaining approximately <0.6% of the volume of normal blood consists of WBCs and Platelets (PLTs). PLTs are small, irregularly shaped anuclear cells that outnumber the WBCs by a factor of ˜30 times. PLTs play a fundamental role in hemostasis and healing by stopping bleeding and releasing a multitude of growth factors that repair and regenerate damaged tissue. However, their adhesive nature interferes with the efficient enrichment and purification of the rare, clinically important target cells. The least prevalent blood cells are WBCs, making up only about one tenth of one percent of the total cells in a typical blood sample. WBCs are critical to the body's immune system, and participate in the defense of the body against infectious disease, foreign materials and hematologic and solid tumor cancers. Nearly all of the cells that are utilized clinically in immunotherapy or regenerative medicine reside within the WBC fraction. WBCs may be further divided into subgroups. The largest and most dense subgroup is the granulocytes (GRNs), which make up approximately 60% of all WBCs. The smaller and less dense subgroup are the mononuclear cells (MNCs), which constitute the remaining approximately 40%. MNCs can further be broken down into lymphocytes and monocytes, but they are collectively referred to as MNCs due to the presence in each cell of a single round nucleus. MNCs are critical elements of the immune system, comprising T cells, B cells and NK cells that migrate to sites of infection in body tissue and then divide and differentiate into macrophages and dendritic cells to elicit an immune response. Many cell therapies now being explored in clinical trials utilize cells that reside within the MNC fraction.
Thus, in order to purify a rare population of cells from blood, bone marrow or leukapheresis, an initial bulk depletion step to remove substantially all of the much more numerous RBCs, GRNs and PLTs to create an enriched MNC fraction is desirable. As previously mentioned, this can be accomplished using centrifugation. Centrifugation is a method of cell processing classified by the FDA as “minimally manipulated”, which provides a simpler regulatory path for FDA clearance. Performing this initial bulk depletion process alone on blood can enrich the rare cell populations residing in the MNC fraction by three orders of magnitude (103), which makes subsequent additional purification and enrichment of the target cells more efficient.
Current methods for isolating target cells begins with a manual method of isolating MNCs require a highly skilled operator working with density gradient mediums, such as Ficoll. Density gradient mediums are small particles of a precise density intermediate to, for instance, the density of granulocytes and MNCs so that when combined and centrifuged, the particles stratify and interpose their layer in between the granulocyte layer and the MNC layer making the subsequent pipette retrieval of the MNCs without the presence of granulocytes more attainable. Subsequent purification to the final target cells within the MNCs requires expensive and complex instrumentation and expensive reagents. These current methods also have low rates of throughput that are unsuitable for purifying rare cells admixed with large quantities of unwanted cells, or have low efficiency of target cell isolation and harvest. These methods may also expose the cells to chemical agents that may have undesirable effects on the cells, or require the use of functionally open systems that present the risk of microbiological contamination of the cells. Therefore, new technologies are needed to enable the isolation, separation, purification, or exchange of medium in which rare cells are suspended with high efficiency, high throughput, and little or no manual intervention, while employing simple equipment, cell-compatible reagents, and functionally closed, sterile systems.